J-HNP-1多肽的重組及體外抗菌作用初步研究
發(fā)布時(shí)間：2018-11-19 19:27
【摘要】：條件致病菌感染病原菌多為耐藥菌,其主要來源于呼吸道、腸道、生殖道等粘膜表面,其感染常見于嚴(yán)重創(chuàng)傷、燒傷、移植術(shù)后、成人免疫缺陷綜合征等重癥患者。目前尚無良好的預(yù)防手段。粘膜上皮細(xì)胞有一個(gè)共同特點(diǎn),即在細(xì)胞膜上具備多聚IgA受體(pIgR)。連有J鏈的IgA分子通過J鏈與pIgR結(jié)合后,將IgA分子轉(zhuǎn)運(yùn)入粘膜細(xì)胞內(nèi)。本研究擬將防御素改建為一種殺菌分子,分子的一端為J鏈,另一端為防御素,稱之為多聚IgA受體(pIgR)配體樣殺菌肽。這種殺菌分子因連有J鏈,具有與pIgR結(jié)合的能力。從而利用pIgR作為橋梁,將防御素轉(zhuǎn)運(yùn)至細(xì)胞內(nèi)的微生物感染處發(fā)揮殺菌作用。本研究將HNP-1(屬α-防御素)與J鏈cDNA連接,然后在哺乳動(dòng)物細(xì)胞系統(tǒng)中表達(dá),并初步檢測其產(chǎn)物體外抗菌活性,為進(jìn)一步研究其細(xì)胞內(nèi)殺菌功能提供依據(jù)。 方法: 應(yīng)用PCR技術(shù)從不同的質(zhì)粒中獲得J鏈基因和HNP-1基因,然后應(yīng)用重組PCR技術(shù)將這兩個(gè)不同的基因連接在一起而成為J-HNP-1基因。將此基因克隆入哺乳動(dòng)物細(xì)胞表達(dá)載體pcDNA3.1(-)/Myc-HisC中。用脂質(zhì)體轉(zhuǎn)染法將此重組子導(dǎo)入COS-7細(xì)胞,分別從mRNA和蛋白質(zhì)水平采用RT-PCR和Western blot分析J-HNP-1的表達(dá)情況,并體外檢測細(xì)胞可溶性蛋白及培養(yǎng)上清的抗菌活性。 結(jié)果: 1.利用重組PCR技術(shù)使J鏈與HNP-1這兩個(gè)不同的基因相連接而成為J-HNP-1重組體。 2.將J-HNP-1重組體克隆入哺乳動(dòng)物表達(dá)載體pcDNA3.1(-)/Myc-HisC質(zhì)粒中,雙重標(biāo)簽基因Myc和6×His位于下游,6×His起著檢測標(biāo)志的作用,使J-HNP-1的表達(dá)產(chǎn)物易于檢測。 3.將重組質(zhì)粒rpcDNA3.1(-)/Myc-HisC/J-HNP-1應(yīng)用正、反方向引物測序,檢測J-HNP-1的DNA序列及鑒定插入的正反方向,獲得正向插入重組子。 4.RT-PCR結(jié)果顯示轉(zhuǎn)染rpcDNA3.1(-)/Myc-HisC/J-HNP-1的COS-7細(xì)胞mRNA
[Abstract]:Most of the pathogenic bacteria are drug-resistant bacteria which mainly come from the mucosal surface of respiratory tract intestinal tract and genital tract. The infection is common in severe patients such as severe trauma burn transplantation adult immunodeficiency syndrome and so on. At present, there are no good preventive measures. A common feature of mucosal epithelial cells is the presence of a poly-IgA receptor (pIgR). On the cell membrane. The IgA molecule with J chain transports IgA molecules into mucosal cells after binding to pIgR via J chain. In this study, defensin was transformed into a bactericidal molecule with a J-chain at one end and a defensin at the other end, which was called a poly (IgA receptor) ligand-like bactericidal peptide (pIgR). This bactericidal molecule has the ability to bind to pIgR because of its J chain. Using pIgR as a bridge, defensins can be transported to microbe infection to play a bactericidal role. In this study, HNP-1 (偽 -defensin) was linked to J chain cDNA, then expressed in mammalian cell system, and its antibacterial activity in vitro was preliminarily detected, which provided the basis for further study of its bactericidal function in cells. Methods: J chain gene and HNP-1 gene were obtained from different plasmids by PCR technique, then the two genes were linked together by recombinant PCR technique to become J-HNP-1 gene. The gene was cloned into mammalian cell expression vector pcDNA3.1 (-) / Myc-HisC. The recombinant plasmid was transfected into COS-7 cells by liposome transfection. The expression of J-HNP-1 was analyzed by RT-PCR and Western blot from mRNA and protein levels, and the antibacterial activity of soluble protein and culture supernatant was detected in vitro. Results: 1. By using recombinant PCR technique, J chain was linked to two different genes of HNP-1 to form J-HNP-1 recombinant. 2. The J-HNP-1 recombinant was cloned into the mammalian expression vector pcDNA3.1 (-) / Myc-HisC. The double label genes Myc and 6 脳 His were located downstream, and 6 脳 His acted as a detection marker, making the expression product of J-HNP-1 easy to detect. 3. The recombinant plasmid rpcDNA3.1 (-) / Myc-HisC/J-HNP-1 was sequenced with positive and reverse primers to detect the DNA sequence of J-HNP-1 and to identify the positive and negative directions of the insertion. 4.RT-PCR results show mRNA of COS-7 cells transfected with rpcDNA3.1 (-) / Myc-HisC/J-HNP-1
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346
本文編號(hào)：2343212
[Abstract]:Most of the pathogenic bacteria are drug-resistant bacteria which mainly come from the mucosal surface of respiratory tract intestinal tract and genital tract. The infection is common in severe patients such as severe trauma burn transplantation adult immunodeficiency syndrome and so on. At present, there are no good preventive measures. A common feature of mucosal epithelial cells is the presence of a poly-IgA receptor (pIgR). On the cell membrane. The IgA molecule with J chain transports IgA molecules into mucosal cells after binding to pIgR via J chain. In this study, defensin was transformed into a bactericidal molecule with a J-chain at one end and a defensin at the other end, which was called a poly (IgA receptor) ligand-like bactericidal peptide (pIgR). This bactericidal molecule has the ability to bind to pIgR because of its J chain. Using pIgR as a bridge, defensins can be transported to microbe infection to play a bactericidal role. In this study, HNP-1 (偽 -defensin) was linked to J chain cDNA, then expressed in mammalian cell system, and its antibacterial activity in vitro was preliminarily detected, which provided the basis for further study of its bactericidal function in cells. Methods: J chain gene and HNP-1 gene were obtained from different plasmids by PCR technique, then the two genes were linked together by recombinant PCR technique to become J-HNP-1 gene. The gene was cloned into mammalian cell expression vector pcDNA3.1 (-) / Myc-HisC. The recombinant plasmid was transfected into COS-7 cells by liposome transfection. The expression of J-HNP-1 was analyzed by RT-PCR and Western blot from mRNA and protein levels, and the antibacterial activity of soluble protein and culture supernatant was detected in vitro. Results: 1. By using recombinant PCR technique, J chain was linked to two different genes of HNP-1 to form J-HNP-1 recombinant. 2. The J-HNP-1 recombinant was cloned into the mammalian expression vector pcDNA3.1 (-) / Myc-HisC. The double label genes Myc and 6 脳 His were located downstream, and 6 脳 His acted as a detection marker, making the expression product of J-HNP-1 easy to detect. 3. The recombinant plasmid rpcDNA3.1 (-) / Myc-HisC/J-HNP-1 was sequenced with positive and reverse primers to detect the DNA sequence of J-HNP-1 and to identify the positive and negative directions of the insertion. 4.RT-PCR results show mRNA of COS-7 cells transfected with rpcDNA3.1 (-) / Myc-HisC/J-HNP-1
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346
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，本文編號(hào)：2343212
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